Luminescence detecting apparatuses and methods

ABSTRACT

A luminescence detecting apparatus and method for analyzing luminescent samples is disclosed. A detecting apparatus may be configured so that light from luminescent samples pass through a collimator, a first lens, a filter, and a camera lens, whereupon an image is created by the optics on the charge-coupled device (CCD) camera. The detecting apparatus may further include central processing control of all operations, multiple wavelength filter wheel, and/or a robot for handling of samples and reagents.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/716,219, filed Mar. 2, 2010, now U.S. Pat. No. 8,278,114, which is adivisional of U.S. application Ser. No. 11/251,873, filed Oct. 18, 2005,now U.S. Pat. No. 7,670,848, which is a divisional of U.S. applicationSer. No. 10/323,669, filed Dec. 20, 2002, now abandoned, which is acontinuation of U.S. application Ser. No. 09/621,961, filed Jul. 21,2000, now U.S. Pat. No. 6,518,068, which claims priority to U.S.provisional application No. 60/144,891, filed Jul. 21, 1999, the entirecontents of each of which applications are hereby incorporated byreference in their entirety for all purposes as if fully set forthherein.

FIELD OF THE INVENTION

This invention relates to the field of apparatus and methods fordetecting and quantifying light emissions, and more particularly, todetecting and quantifying light emitted from luminescent-based assays.Still more particularly, this invention pertains to apparatus andmethods for detecting and quantifying luminescence such asbioluminescence and/or chemiluminescence from luminescent assays as anindicator of the presence or amount of a target compound. Preferredembodiments of the invention include as an imaging device a chargecoupled device (CCD) camera and a computer for analyzing data collectedby the imaging device. Further preferred embodiments include thecapacity for use in high throughput screening (HTS) applications, andprovide for robot handling of assay plates.

DESCRIPTION OF THE RELATED ART

The analysis of the luminescence of a substance, and specifically theanalysis of either bioluminescence (BL) or chemiluminescence (CL), isbecoming an increasingly useful method of making quantitativedeterminations of a variety of luminescent analytes.

Recently, methods have been introduced that utilize luminescencedetection for quantitatively analyzing analytes in an immunoassayprotocol. Such luminescence immunoassays (LIA) offer the potential ofcombining the reaction specificity of immunospecific antibodies orhybridizing nucleic acid sequences and similar specific ligands with thehigh sensitivity available through light detection. Traditionally,radioactive reagents have been used for such purposes, and thespecificity and sensitivity of LIA reagents is generally comparable tothose employing traditional radiolabelling. However, LIA is thepreferred analytical method for many applications, owing to the nontoxicnature of LIA reagents and the longer shelf lives of LIA reagentsrelative to radioactive reagents.

Among other luminescent reagents, chemiluminescent compounds such as1,2-dioxetanes, developed by Tropix, Inc. and other stablechemiluminescent molecules, such as xanthan esters and the like, are incommercial use. These compounds are triggered to release light throughdecomposition triggered by an agent, frequently an enzyme such asalkaline phosphatase, which is present only in the presence, or specificabsence, of the target compound. The detection of light emission is aqualitative indication, and the amount of light emitted can bequantified as an indicator of the amount of triggering agent, andtherefore target compound, present. Other well known luminescentcompounds can be used as well.

Luminescent release may sometimes be enhanced by the presence of anenhancement agent that amplifies or increases the amount of lightreleased. This can be achieved by using agents which sequester theluminescent reagents in a microenvironment which reduces suppression oflight emission. Much biological work is done, perforce, in aqueousmedia. Water typically suppresses light emission. By providingcompounds, such as water soluble polymeric onium salts (ammonium,phosphonium, sulfonium, etc.) small regions where water is excluded thatmay sequester the light emitting compound may be provided.

The majority of instrumentation used to monitor light emitting reactions(luminometers) use one or more photomultiplier tubes (PMTs) to detectthe photons emitted. These are designed to detect light at the low lightlevels associated with luminescent reactions. The rate at which a PMTbased microplate luminometer can measure signal from all wells of theplate is limited by the number of PMTs used. Most microplateluminometers have only one PMT so a 384 well plate requires four timeslonger than is required to read a 96-well plate.

The nature of biological research dictates that numerous samples beassayed concurrently, e.g., for reaction of a chemiluminescent substratewith an enzyme. This is particularly true in gene screening and drugdiscovery, where thousands of samples varying by concentration,composition, media, etc. must be tested. This requires that multiplesamples be reacted simultaneously, and screened for luminescence.However, there is a need for high speed processing, as thechemiluminescence or bioluminescence may diminish with time.Simultaneously screening multiple samples results in improved datacollection times, which subsequently permits faster data analysis, andcontingent improved reliability of the analyzed data.

In order for each specific sample analyte's luminescence to be analyzedwith the desired degree of accuracy, the light emission from each samplemust be isolated from the samples being analyzed concurrently. In suchcircumstances, stray light from external light sources or adjacentsamples, even when those light levels are low, can be problematic.Conventional assays, particularly those employing high throughputscreening (HTS) use microplates, plastic trays provided with multiplewells, as separate reaction chambers to accommodate the many samples tobe tested. Plates currently in use include 96- and 384-well plates. Inresponse to the increasing demand for HTS speed and miniaturization,plates having 1,536 wells are being introduced. An especially difficultimpediment to accurate luminescence analysis is the inadvertentdetection of light in sample wells adjacent to wells with high signalintensity. This phenomenon of light measurement interference by adjacentsamples is termed ‘crosstalk’ and can lead to assignment of erroneousvalues to samples in the adjacent wells if the signal in those wells isactually weak.

Some previously proposed luminometers include those described in U.S.Pat. No. 4,772,453; U.S. Pat. No. 4,366,118; and European Patent No. EP0025350. U.S. Pat. No. 4,772,453 describes a luminometer having a fixedphotodetector positioned above a platform carrying a plurality of samplecells. Each cell is positioned in turn under an aperture through whichlight from the sample is directed to the photodetector. U.S. Pat. No.4,366,118 describes a luminometer in which light emitted from a lineararray of samples is detected laterally instead of above the sample.Finally, EP 0025350 describes a luminometer in which light emittedthrough the bottom of a sample well is detected by a movablephotodetector array positioned underneath the wells.

Further refinements of luminometers have been proposed in which a liquidinjection system for initiating the luminescence reaction just prior todetection is employed, as disclosed in EP 0025350. Also, a temperaturecontrol mechanism has been proposed for use in a luminometer in U.S.Pat. No. 4,099,920. Control of the temperature of luminescent samplesmay be important, for example, when it is desired to incubate thesamples at an elevated temperature.

A variety of light detection systems for HTS applications are availablein the market. These include the LEADseeker™ from Amersham/Pharmacia,the ViewLux™ offered by PerkinElmer and CLIPR™ from Molecular Devices.These devices are all expensive, large dimensioned (floorbased models),exhibit only limited compatibility with robotic devices for platepreparation and loading, have a limited dynamic range, and/or useoptical detection methods which do not reduce, or account for,crosstalk. The optical systems used are typically complex teleconcentricglass lens systems, which may provide a distorted view of wells at theedges of the plates, and the systems are frequently expensive, costingin excess of $200,000.00. Perhaps the most popular detection apparatusis the TopCount™, a PMT-based detection system from Packard. Althoughthe TopCount™ device has a desirable dynamic range, it is not capable ofreading 1,536 well plates, and it does not image the whole platesimultaneously.

Crosstalk from adjacent samples remains a significant obstacle to thedevelopment of improved luminescence analysis in imaging-based systems.This can be appreciated as a phenomenon of simple optics, whereluminescent samples produce stray light which can interfere with thelight from adjacent samples. Furthermore, the development ofluminometers capable of detecting and analyzing samples with extremelylow light levels are particularly vulnerable to crosstalk interference.

SUMMARY OF THE INVENTION

In order to meet the above-identified needs that are unsatisfied by theprior art, it is a principal object and purpose of the present inventionto provide a luminescence detecting apparatus that will permit theanalysis of luminescent samples. It is a further object of the presentinvention to provide a luminescence detecting apparatus capable ofsimultaneously analyzing a large number of luminescent samples. In apreferred embodiment of the present invention, a luminescence detectingapparatus is provided that simultaneously analyzes multiple samples heldin wells, where the well plates contain as many as 1,536 wells. Thepresent invention further includes robot handling of the multiple welltrays during analysis.

It is yet another object of the present invention to provide aluminescence detecting apparatus capable of analyzing low light levelluminescent samples, while minimizing crosstalk from adjacent samples,including and especially minimizing crosstalk from adjacent samples withhigher light level output than the sample to be analyzed.

The apparatus of this invention employs a Fresnel lens arrangement, witha vertical collimator above the well plate, with dimensions to match thenumber of wells. Thus, a 1,536-well plate will employ a dark collimatorabove the plate with 1,536 cells in registry with the wells of theplate. Fixed above the collimator is a Fresnel lens, which refracts thelight such that the view above the lens appears to be looking straightdown into each well, regardless of its position on the plate, even atthe edges.

Above the Fresnel lens is a CCD camera arranged so as to take the imageof the entire plate at one time, viewing through a 35 mm wide anglelens, to give whole plate imaging on a rapid basis. Between the CCD andFresnel/collimator is a filter, typically arrayed on a filter wheel,disposed at an angle to the lens. The filter is selected to permit thepassage of the specific wavelength of the light emitted, and reflect orabsorb all others. Several filters may be provided on the wheel, topermit sequential detection of light emitted from multiple reagentsemitting light at different wavelengths.

The samples are fed to the optical detection platform through a loadingdevice designed to work well with robotic and automated preparationsystems. The well-plate, with reaction mixture already provided, isplaced on a shuttle by a human, or preferably, robot. Alignment of theplate on the shuttle may be relatively coarse, notwithstanding therequirement for tight tolerances to match the collimator grid array. Asthe shuttle leaves the loading position, a resilient means urges theplate into strict conformal alignment. The shuttle positions the plateunder an overhead injection bar, which may accommodate up to sixteenwells in a column at one time. If not previously added, a triggeringagent or luminescent reagent is added to the sample wells, and the plateindexes forward to load the next column of wells across the plate. Theshuttle then advances through a door into the sample chamber, and theplate is aligned with the collimator and the Fresnel lens. Since manyreactions proceed better, or only, at elevated temperatures, the samplechamber is insulated, and provided with heating means, for heating theair in or provided to the chamber. In order to maintain temperature inthe chamber close to room temperature and to accurately controltemperature, the chamber may also be provided with a heat exchanger.

The light emission from the entire multiple well plate is imaged atonce, with subsequent imaging through a different filter if multiplewavelengths are employed. The signal obtained is processed to furtherreduce crosstalk reduced by the collimator and the presence and amountof luminescence is quickly detected and calculated by a personalcomputer using automated software. Data is then reported as intensityper well or further analyzed relative to specific assay standards.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a cross section of a preferred embodiment of a luminescencedetecting apparatus according to the present invention;

FIG. 2 is a detailed cross section of the optics of a luminescencedetecting apparatus according to the present invention.

FIG. 3 is a cross-sectional view of the plate transport system of theinvention.

FIG. 4 is a perspective illustration of the injector arm assembly of theinvention.

FIG. 5 is an exploded view of the filter wheel assembly.

FIG. 6 is a cross-sectional view of the optical housing.

FIG. 6A is a plan view of a robotic mechanism of the invention.

FIG. 7 is a flow chart illustration of the processing method of theinvention.

FIGS. 8-15 are illustrations of the results obtained using the inventionin Examples 1-10, respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring now to the drawings, wherein like reference numerals designateidentical or corresponding parts throughout the several views, and moreparticularly to FIG. 1 thereof, a preferred embodiment of theluminescence detecting apparatus of the present invention uses a shuttleor tray to carry a micro plate (plate) 10 comprising a plurality ofsample wells 20 which may in the preferred embodiment number as many as1,536 or more. Persons of ordinary skill in the relevant art willrecognize that the number of sample wells 20 is limited only by thephysical dimensions and optical characteristics of the luminometerelements, and not by the technology of the present invention. The samplewells 20 may be filled with analyte manually, or robotically prior todelivery to the inventive apparatus. Agents necessary forchemiluminescence may be filled automatically via the injector 30, towhich analyte is supplied through an array of supply tubes 40 or priorto placing the plate on the tray. Typically, the sample wells willcontain chemiluminescent reagents. These reagents emit light atintensities proportional to the concentration of analyte in the sample.This light can be very low intensity and requires an instrument withsufficient sensitivity to achieve the desired detection limits.

The operation of injector 30 is controlled by central processor 50,which in the preferred embodiment may control the operation of allelements of the luminometer of the present invention. Data collection,analysis and presentation may also be controlled by processor 50.Further in a preferred embodiment of the present invention, the injector30 may also be used to add buffer solutions to the analytes and also toadd reagents that enable “glow” and/or “flash” luminescence imaging,that is sustained or brief, intense emission, respectively, all undercontrol of central processor 50.

After the analytes are placed in the sample wells 20, plate 10 is placedin sample chamber 55, which is located in optical chamber 60 at a fixedfocal distance from and directly under the charge-coupled device (CCD)camera 70, in order to permit the CCD camera to image the luminescentsample accurately. The sample chamber 55 is preferably capable ofprecise temperature control, as many luminescent reagents and specificluminescent reactions are temperature dependent. Temperature control isprovided by central processor 50, which can vary the temperature foreach individual sample plate 10, as central processor 50 controls themovement and injection of the sample wells 20 in each sample tray 10. Ina preferred embodiment of the luminometer of the present invention,central processor 50 also controls an industrial robot (not shown) whichperforms the activities involving analyte handling in the luminometer ofthe present invention.

With the plate 10 placed in the sample chamber 55, the optics 80 deliverthe image of the complete microplate 10 as a single image to the CCDcamera 70.

Although the operation of the luminometer of this invention is anintegral, continuous practice, and all elements of the luminometercooperate together to provide precise, accurate and reliable data, theinvention may be more easily understood by reference to three separate,integrated systems, the optics system, the mechanical system and theprocessing system. Each is discussed in turn, with a discussion ofexamples of the operation as a whole to follow.

Optics System

Turning now to FIG. 2, the optics 80 are shown in further detail.Luminescent emission 100 from the analyte in plate well 20 located inthe plate 10 travels first through dark collimator 110, which permitsonly parallel and semi-parallel light rays to exit the sample wells 20for eventual imaging by the CCD camera 70. The effect of collimationassists with the prevention of stray light from the sample wells 20 andwith the elimination of crosstalk between luminescent samples. Thecollimator 110 may be sealably engaged, or in close proximity, to thesample tray 10, to enhance the restriction of stray light from thesamples. Each well 10 is in strict registration and alignment with acorresponding grid opening in collimator 110. From the collimator 110,the luminescent radiation passes through a Fresnel field lens 120, whichfocuses the light toward filter 130. In a preferred embodiment of thepresent invention, the collimator 110 and Fresnel field lens 120 arepackaged in a cassette that can be changed by the user. Such anequipment change may be necessitated by varying optical characteristicsof different analytes and different well distributions in plates.

The use of a Fresnel field lens is preferable to alternative opticaldevices for several reasons. Initially, improvements in design andmaterials have capitalized on the superior optical capabilities of theFresnel lens, while virtually eliminating its once inherent limitations.Today, many Fresnel lenses are made of molded plastic, creating analmost flawless surface with very little scatter light. The eliminationof scatter light is an important element of eliminating crosstalkbetween adjacent samples in the luminometer of the present invention.Furthermore, improved types of plastics commonly employed in themanufacturing of Fresnel lenses and other optical devices have opticalqualities equivalent to ground glass lenses.

Using high tech processes such as computer-controlled diamond turning,complex aspheric surfaces can be cut into a long lasting mold forcasting Fresnel lenses. In this manner, Fresnel lenses can bemanufactured to produce the precise optical imaging effect that is mostefficient for a charge coupled device camera, as in the presentinvention. Also, Fresnel lenses offer an advantage over conventionallenses in that they can be molded flat and very thin. Because of theshape of the Fresnel lens, it can easily be integrated directly into thehousing of the luminometer, enhancing the light-tight propertiesnecessary for accurate imaging of low light samples. Furthermore,Fresnel lenses are much less expensive than comparable conventionalglass lenses.

As with any other lens, the total beam spread from a Fresnel lensdepends on the size of the source in relation to the focal length of thelens. Smaller sources, such as luminescent assay samples, and longerfocal lengths produce more compact beams. Since there are practicallimitations to minimizing the geometry and dimensions of the optics 80in the luminometer of the present invention, the use of Fresnel fieldlens 120 provides the greatest opportunity for fine-tuned optics. Theemissions from plate 10 pass through lens 120, and are refracted suchthat the image obtained at CCD 70 appears to look directly downward intoall wells, even laterally displaced (edge) ones. This feature istypically called “telecentric.”

Further in a preferred embodiment of the present invention, the filter130 may be configured on a wheel, wherein different filter elements mayoccupy different portions of the wheel, depending on the luminescentcharacteristics of the sample being analyzed. Filter 130 is preferablyinclined at an angle of 20°-30° relative to the CCD, so that strayreflected light is reflected outside the field of view. Specifically,the filter wheel 130 permits the selection of different wavelengthranges, which not only permit high quality imaging, but may be used toseparate the emissions of different reagents emitting at differentwavelengths. Again, the filter wheel 130 is controlled by centralprocessor 50, in coordination with central processor 50's control of theindividual sample wells 20 in the sample plate 10. In many assays, suchas those addressed in pending U.S. patent application Ser. No.08/579,787, incorporated by reference herein, multiple luminescentreagents, which emit at different wavelengths, are employed in a singlewell. Using multiple filters, each can be imaged in turn, and the trueconcentration can be calculated from the data set resulting usingpre-stored calibration factors. Filter 130 is preferably provided withan infrared (IR) filter operating in conjunction with the selectedbandpass, or as an independent element. Applicants have discovered thatstray IR radiation, resulting from the plate phosphorescence, resultingin abnormally high backgrounds. An IR filter suppresses this.

From the filter wheel 130, the sample-emitted light passes throughcamera lens 140, which in the preferred embodiment is a large aperture,low distortion, camera lens. Camera lens 140 focuses the image of thesample on the CCD chip 70. In the preferred embodiment of the presentinvention, CCD camera 70 is a cooled, low noise, high resolution device.The lens is preferably a 35 mm wide angle lens with a low light level (F1.4) large aperture character. Magnification of 3-6, preferably about5.5, is preferred. In preferred embodiments CCD camera 70 is providedwith an anti-blooming CCD chip, to enhance dynamic range, which is about10⁵ in the claimed invention, referred to as the NorthStar™ luminometer.Blooming occurs when a single pixel is overloaded with light and itsphotoelectrons overflow the CCD device well capacity, obliteratingsurrounding pixels. Further in the preferred embodiment of theluminometer of the present invention, the selected CCD camera includes aliquid cooled thermoelectric (Peltier) device providing cooling of theCCD to approximately −35° C., and the CCD has 1280×1024 pixels, each ofwhich are 16 μm square, producing a total active area of 20.5 mm×16.4mm. The quantum efficiency averages 15% over the range from 450nanometers to 800 nm. The output is digitized to 16 bit precision andpixels can be “binned” to reduce electronic noise.

By using the features disclosed herein, the luminometer of the presentinvention has a spatial resolution capable of providing high qualityimaging of high density, sample trays. The noise performance and CCDtemperature are designed to provide the desired detection limit.

Mechanics

The mechanical systems of the luminometer workstation of this inventionare designed to achieve automated, high throughput precise delivery ofmicroplates in registration with a collimator 110 so as to be read bythe CCD Camera 70. To this end, as shown in FIG. 3, a cross-section ofthe inventive luminometer shuttle 200 translates from a load position202, where plates 10 are loaded on to the shuttle, preferably by arobotic device such as robot arm, and the shuttle 200 then translatestowards sample chamber 55, to read position 203. Shuttle 200 is causedto translate by a conventional stepper motor (not pictured). As shuttle200 advances toward sample chamber 55, it may stop underneath injector30. Injector 30 is more fully illustrated below in FIG. 4. Referringstill to FIG. 3, injector 30 delivers fluid reagents drawn fromreservoir 204. Syringe pump 205 draws the fluid reagents from reservoir204, and pumps the fluid to the injector tubes 40. Two way valve 206controls the passage of the fluid drawn by syringe pump 205 fromreservoir 204 and pumped by syringe pump 205 to the supply tubes 40. Inactual practice, there are as many injector tubes 40 as injection portsbeing used, and multiple syringe pumps 205 are also used. As will beshown below in FIG. 4, injector 30 has up to sixteen injection ports302. The plates used in conjunction with the luminometer when injectionis used are typically prepared with up to sixteen wells in a column. Asthe shuttle 200 advances plate 10 underneath injector 30, shuttle 200stops so that the first column 208 of wells is directly aligned underinjector 30. Precise amounts of analyte are delivered to the first setof wells, and shuttle 200 indexes forward one column, so as to injectreagent into the second column of wells 210. This process is repeateduntil all wells are filled. Thereafter, shuttle 200 advances forwardinto sample chamber 55 through hinged door 212. In the alternative, door212 may be a guillotine door or similar type of closing mechanism. Thewells of plate 10 are then read in sample chamber 55. Upon completion ofreading, shuttle 200 translates back to load position 202.

Before shuttle 200 advances to the injection bar, it may be necessary tofully prime the tube with fluid, so as to provide for precise deliveryinto the plate. Trough 304 swings out from its storage position parallelto the direction of travel of shuttle 200, shown by an arrow, to aposition directly underlying the injector 30, perpendicular to thedirection of travel. Fluid in the injector and tubes 204 are deliveredinto trough 304, and removed by suction. Trough 304 then returns to itsrest position, parallel to, and away from, the direction of travel ofthe shuttle 200, when the shuttle is moved toward the sample chamber 55.On its return trip to load position 202, locator 214 on shuttle 200 isengaged by cam 216. Locator 214 is mounted on a resilient means, suchthat when engaged by cam 216, the locator 214 recesses away from plate10. This permits removal of plate 10, and delivery from a robotic arm orother source of a fresh plate 10, without the requirement of preciselocation. As shuttle 200 moves away from load 202, locator 214 is urgedforward, firmly locating plate 10 in place. Plate 10 is held againstshoulder 217 by the resilient urging of locator 214.

It is important that each plate be precisely identified, so that resultsare correlated with the correct test samples. In most HTS laboratories,most microplates are labeled with a unique “bar code.” The label isoften placed on the surface perpendicular to the plane of the plateitself. To permit precise identification of each plate, a bar codereader 218 is mounted on the luminometer housing generally indicated at299 and directly above the door 212, for example on an arm or flange220. Bar code reader 218 is focused on a mirror 222 which in turnpermits reading directly off the front or leading edge of plate 10 as itapproaches on shuttle 200. Thus, before each plate arrives in the samplechamber, its identity has been precisely recorded in processor 50, andthe results obtained can be correlated therewith. Persons of ordinaryskill in the art will recognize that a variety of configurations ofalignment and placement of both bar code reader 218 and mirror 222 willresult in the desired identification.

As more clearly shown in FIG. 4, injector 30 may be precisely located byoperation of actuator wheel 306, provided with positions correspondingto the total number of wells on the plates being assayed. Similarly, thevertical position, to account for the different thicknesses of theplate, may be controlled by wheel 308. Given the simple translationmovement of shuttle 200, and the precise locating and identification ofeach plate carried, rapid cycling of micro-plate test plates into andout of sample chamber 55 can be effected.

As described above in connection with the optics system of theinvention, a filter is provided which includes or reflects passage oflight other than light falling within the selected wavelength of theluminescent emitter in use. The filter assembly is illustrated inexploded format in FIG. 5. Filter frame 502 is supported by arm 504which is connected to the hub of the filter wheel 506. Multipledifferent filters may be provided on a single wheel. The filter itself,508, is securely mounted on the frame and held there by cover 510, whichis secured to frame 502 by grommets, screws or other holding devices512. As noted, filter wheel is positioned so as to hold filter 508 inframe 502 at in incline with respect to collimator 110, of about 22°nominally, so as to direct any reflections outside the field of view.Light passes through the filter opening 514, in alignment with cameralens 140 and CCD camera 70. As further noted above, filter 508preferably includes an infrared block, either as a component of thefilter itself, or as a component provided in addition to the filter forthe measured light. An IR block is of value to prevent infraredemissions caused by extraneous radiation from altering the imagereceived by the CCD camera.

Optical chamber 60 is more fully illustrated in FIG. 6. As shown,optical chamber 60 is bounded by optical housing 602 in which fitssample housing 604. When a plate 10 is loaded into optical chamber 60,the plate is secured in sample housing 604 which is positioned inregistry with collimator 110, over which is provided Fresnel lens 120.While many luminescent assays can be provided at ambient temperatures,some require elevated temperatures. The luminometer of this device isprovided with a sample chamber in which the sample housing 604 carriesinsulation 606 which, in a preferred embodiment is polyurethane foam,and heater element 608 to raise the temperature in the sample chamber 55above ambient temperature, up to about 42° C.

There is a tendency, even at ambient conditions, for condensation tocollect on the surface of the Fresnel lens 120, as a result of moisturecoming from the filled wells of plate 10. The defogger 610 directs astream of air heated just a few degrees, preferably about 2-3° degrees,above ambient conditions, or above the temperature of the chamber if thechamber is above ambient conditions, across the surface of the Fresnellens 120, effectively preventing condensation. Mounted at the top of theinterior of optical chamber 60 is filter motor 610 which drives filterwheel 612, on which may be mounted filters 614 of varying wavelength,for filtering undesirable wavelengths prior to imaging. Of course, aregion is provided, indicated at 616, in the optical housing 602 of theoptical chamber 60 for light to be directed onto the CCD camera afterpassing through the filter 614. The dimensions of optical chamber 60 areexaggerated in FIG. 6 to illustrate the relationship between the opticalchamber 60 and the filter wheel 612, and defogger 610. In practice, thefilter is located inside the optical chamber 60, and outside the samplehousing 604 but alternate locations are possible while still achievingthe desired function.

In FIG. 6A, a plan view of a novel robotic mechanism 616 is displayed ina preferred embodiment of the present invention, which provides capacityfor use in high throughput screening (HTS) applications. Referring toFIG. 6A, the operation is as follows: robot plate stacks 620, 622, 624,626, and 628 each can be filled with multiple sample plates 10, arrangedin a vertical stack. In the preferred embodiment of FIG. 6A, robot platestack 628 is designated as the discard stack. The remaining robot platestacks 620, 622, 624, and 626 can be programmed in order of delivery bysoftware controlled by processor 50 (not shown). In order to load orpick plates from any of these stacks, robot arm 630 moves vertically androtationally to the desired robot plate stack, under control of thesoftware programmed in processor 50.

When commanded by processor 50, transport 200 of the instrument willmove the sample plate 10 from load position 202 to the Read position203, and return it to load position 202 when imaging is complete. In theembodiment of the invention shown in FIG. 6A, the elapsed time betweenmoving the sample plate 10 from load position 202 to the read position203, and returning it to load position 202 is typically 30-120 seconds,including imaging time.

Staging positions 632 and 634 are located at 45 degree positionsrelative to the position of robot arm 630. In one embodiment, whileimaging is in process, the robot arm 630 can place a sample plate 10 atstaging position 632, in preparation for placing the sample plate 10 inload position 202. When the imaging is complete, the robot can move theread plate from load position 202 to staging position 634, then load theplate from staging position 632 to load position 202, and while thesample plate 10 is being imaged, the robot can move the plate fromstaging position 634 to the discard stack 628, and place a new sampleplate 10 at staging position 632. In practice, the staging positions areat approximately the same level as the load position, so movement isvery quick. In the preferred embodiment, the robot arm 630 can do thetime consuming moves to any of robot plate stacks 620, 622, 624, and 626while imaging is going on, rather than in series with imaging.

With the staging positions 632 and 634, the cycle time for a singlesample plate 10 is 2 moves from/to staging areas (3 seconds each), plus2 transport moves IN/OUT to read position 203 (3 seconds each), plus theintegration time (image exposure) time (typically 60 seconds), for atotal cycle time of 72 seconds. Without using staging positions 632 and634, the time would be 2 moves to stacks (30 seconds each), plus 2transports (3 seconds each), plus the integration time (typically 60seconds) for a total of 126 seconds. As described in the preferredembodiment of the robotic mechanism 616, the use of staging positions632 and 634 decreases cycle time by 43%.

Processing

As set forth above, the mechanical and optical systems of theluminometer workstation of the invention are designed to provideprecise, quantified luminescent values in an HTS environment, takingadvantage of the use of a Fresnel lens/collimator assembly to permitsingle image viewing by the CCD camera, and subsequent analysis. Thecollimator, the lens and the camera together combine to reducecross-talk experienced in prior art attempts. The signals obtained arefurther processed, as illustrated in FIG. 7, through software loadedonto processor 50, or other convenient method, to further refine thevalues obtained.

Prior to processing image data collected through the integratedmechanical and optical systems of the invention herein described, theintegrated processing component of the invention must first control themechanical alignment of those integrated mechanical and optical systemsfor reliable data collection. This process is conducted under control ofthe processor 50. To conduct an alignment test, the luminescencedetection of the present invention measures the light emitted from fourtest sample wells, called hot wells, of a test plate. In a preferredembodiment, the hot wells are located near each corner of the sampletray used for the alignment testing. The adjacent well crosstalk fromeach of the four hot wells is analyzed, and the values are compared.When the collimator is aligned precisely over the sample well tray, thecrosstalk values will be symmetrical for the four hot wells. Thesoftware of the present invention flags any errors detected, such asincorrect number of test sample wells, incorrect intensity, or incorrectlocation. After the detection of no errors or after the correction ofdetected and flagged errors, the software of the present inventionperforms a symmetry calculation to determine precise alignment of thesample well tray, collimator, Fresnel lens and CCD camera assembly. In aknown embodiment of the invention, known software techniques areemployed to perform the symmetry calculation process by performing thefollowing steps:

-   1. Extract the hot well and vertical and horizontal adjacent well    intensities;-   2. Calculate the averages of the horizontal and vertical adjacent    well intensities separately for each hot well;-   3. Calculate the differences between the actual adjacent intensity    vs. the average for each of the horizontal and vertical directions;-   4. Normalize the differences by the hot well intensity to convert to    a percentage intensity value;-   5. Find the worst case absolute value of the differences and display    that as the overall misalignment;-   6. Calculate the average X-direction (horizontal) misalignment by    averaging the four adjacent wells to the right (horizontal    direction) of the hot wells;-   7. Calculate the average Y-direction (vertical) misalignment by    averaging the four adjacent wells to the top (vertical direction) of    the hot wells;-   8. Calculate the rotational misalignment by averaging the left side    hot well vertical adjacent wells at the top of the hot wells, and    subtracting that from the average of the right side hot well    vertical adjacent wells, thereby indicating any tilt in adjacent    well values.

In step A, three actual images for each filter/emitter are taken. A₁ isa precursor image, A₂ is the full integration time image, and A₃ ispost-cursor image. The precursor and post-cursor images are taken toavoid the problem of pixel saturation and to extend the detectiondynamic range. The precursor and post-cursor images refer to reducedintegration time images, which should not contain multiple saturatedpixels. If more than six pixels of the full integration time image aresaturated, the pre- and post-cursor images are averaged together to formthe actual data for that well area. In the absence of six pixelsaturation, the full integration time image is used.

In order to clearly isolate and read each pixel, in step B, each imageis subjected to edge detection and masking, a processing step wherebythe edge of each well or corresponding light image is identified, orannotated, to set off and clearly separate each well region of interest,as disclosed in U.S. patent application Ser. No. 09/351,660,incorporated herein by reference. Again, edge detection and masking isperformed for each of B₁, B₂ and B₃, referring to the pre-cursor, fullintegration time image and post-cursor images, respectively. The imagesare then subjected to “outlier” correction, correcting or “shaving”outliers and anomalies. In this process, the pixels within the region ofinterest are examined to identify “outliers”—those that are in grossdisagreement with their neighbors, in terms of light intensity detected,and if the intensity of a given pixel or small pixel area issignificantly different than neighboring pixels or pixel areas, then theaverage of the surrounding pixels or areas is used to replace erroneousdata. This can be due to random radiation, such as that caused by cosmicrays. In this process, this type of intensity is corrected.

Subsequently, in step C, each image C₁, C₂ and C₃ is subjected to darksubtraction, subtracting the dark background, so as to obtain averagepixel values within each mask-defined region of interest. Thesubtraction is done on a well-by-well basis from stored libraries whichare updated periodically.

Specifically, the dark subtraction is conducted to correct for the factthat even in the absence of light, CCD cameras can output low levelpixel or bin values. This value includes the electronic bias voltage,which is invariant of position and integration time, and the “darkcurrent,” which may vary by position, and is proportional to integrationtime and to the temperature of the CCD. The CCD may also have faultypixels that are always high level or saturated regardless of lightinput.

The processing software of the invention subtracts this background imageor data from the real sample well image data in step C. As persons ofordinary skill in the relevant art will recognize, it is known to take a“dark” image immediately before or after a real image, imaging for thesame integration time in both cases, and subtracting the “dark” imagedata from the real image data. In the preferred embodiment of theinvention, “dark” image data is collected intermittently, preferably atspecific time intervals. The initial “dark” image background data iscollected at startup, and then typically at four hour intervals duringimage processing operations.

Because the background image has an integration time-invariant componentand an integration time-variant component, data is collected for eachsample well at minimum integration time and at maximum integration time,and a “slope/intercept” line is calculated between the two data points,using known data analysis techniques. This calculation permits datainterpolation for any integration time between the minimum and maximum,and also permits data extrapolation for integration times below orbeyond the minimum and maximum integration times.

In a preferred embodiment of the invention, a CCD camera is employedthat has two separate “dark” current functions, caused by the CCD outputamplifier. Operation of the amplifier generates heat and necessarilycreates background “dark” image data. In the preferred embodiment, forintegration times of less than 10 seconds, the amplifier operatescontinuously, whereas for integration times of more than 10 seconds, theamplifier remains off until immediately prior to the read operation. The“slope/intercept” line calculated for integration times of more than 10seconds will then necessarily have a lower slope than a“slope/intercept” line calculated for integration times of less than 10seconds. In step C, the processing software element allows separatecollection and least squares regression for both the 0 to 10 secondintegration time region a processor 50, the “dark” background image datais stored separately for each individual AOI.

“Dark” current and bias can also vary over time. The processing softwareelement corrects for this effect by comparing the integration timenormalized (using the regression line technique described above) “darkreference” pixel values (outside the imaging field-described above),that were taken when the “dark” background images were taken, versus the“dark reference” pixel values taken while real sample well images arebeing taken. The difference between the values is then subtracted oradded, as applicable, as a global number, to the “dark” background data.This corrects for bias drift and also for global CCD temperature drift.

As mentioned, all of the above “dark background”interpolation/subtraction of step C is done on a well by well basis.

At step D, if pixel saturation has occurred such that the average of thepre-cursor and post-cursor image must be used, the image data ismultiplied by the reciprocal of the percentage represented by thepre-cursor images (e.g., 3%).

In step E, the well data is corrected for uniformity variations using acalibration file that is the reciprocal of the system response to aperfectly uniform input illumination.

In step F, the cross-talk correction is effected by processing the dataas a whole and preparing a final image in much the same fashion asreconstruction of three dimensional images from a two dimensional dataarray is practiced.

Specifically in a preferred embodiment of step F, the impulse responsefunction (IRF) is collected for all 96 wells of the 96 well plate type.This is done by filling one particular well in a given plate with a highintensity luminescent source, imaging the plate, and analyzing all ofthe wells in the plate for their response to the one high intensitywell. The IRF is collected for all of the wells individually byrepeating the process for every different well location desired for thecomplete data set. For 384 plate types, 96 sampling areas are selected,and data for the wells in between the selected sampled areas areinterpolated in two dimensions. In the preferred embodiment, the 96sampling areas comprise every second row and every second column,starting at the outside and working toward the center. Because in the384 well plates the number of rows and columns is even, the two centerrows and the two center columns are interpolated. The reflections in a384 well plate are also modeled, and used to predict and interpolatereflections for the missing input data. Further in the preferredembodiment, all wells are normalized to the well with the highestintensity.

Subsequently in step F, the two-dimensional array of well IRF values foreach welfare “unfolded” into a one-dimensional column array, and thetwo-dimensional arrays of IRF values for other wells are added assubsequent columns, as shown in Chart 1 following:

CHART 1 Unfolded Data Into Column 1 IRF for IRF for IRF for A1 B1 C1 A1A1 A1 Etc B1 B1 B1 C1 C1 C1 D1 D1 D1 E1 E1 E1 F1 F1 F1 G1 G1 G1 H1 H1 H1A2 A2 A2 B2 B2 B2 C2 C2 C2 Etc Etc Etc

The unfolded matrix, which has the form of an N×N matrix, where N=thenumber of wells to be corrected, comprises a full characterization ofthe instrument crosstalk, including reflection factors. This unfoldedmatrix is then inverted, using known matrix inversion techniques, andused as a correction to matrix multiply a one-dimensional matrixunfolded from real assay data. This arithmetic process may be shown asmatrix algebra:[true source distribution]×[system IRF]=[instrument output] solving for[true source distribution] produces[true source distribution]={1/[system IRF]}×[instrument output]

Subsequently, the calculated well intensities resulting from the aboveprocessing are calibrated to an absolute parameter of interest, such asthe concentration of a known reporter enzyme. This calibration isconducted through a normalization process producing any of a variety ofcalibration curves, which will be familiar to those of ordinary skill inthe relevant art.

In optional step G, the processed image information is subjected to anynecessary post adjustment processing, for appropriate correlation withthe materials tested. Specifically, in a preferred embodiment, theprocessing software of the present invention is capable of performingmulti-component analysis. The basic problem is to calculate separatelythe concentration of a single reagent in a single sample containingother different reagents. Typically, the reagents used with theinvention are formulated so as to emit over different, but perhapsoverlapping, spectrums. As earlier described with respect to theintegrated optical element, the first step of separating the light frommultiple reagents is accomplished by optical bandpass filters, which aredesigned to maximize the sensitivity of the target reagent emission,while minimizing the sensitivity to other non-target reagent emission.In the present embodiment of the invention, there is one optical filterfor each target reagent emission spectrum.

Since optical filters are interference devices, their bandpasscharacteristics vary, dependent on the angle of incidence of theemission to be filtered. The angle of incidence will be unique for eachwell because each well's specific location is unique relative to theoptical filter. Accordingly, all calculations and filter coefficientsmust be unique per sample well. The multi-component calibration isperformed as follows:

Prior to the real multiplexed (multiple reagent) samples, standardscontaining only a single reagent in each well are imaged and analyzed.These standards will produce a set of coefficients to be usedcollectively as multi-component coefficients for each optical filter,for each well. For a given optical filter, the target reagent for thatfilter should produce the highest output. The other reagents may alsohave spectra in the filter's bandpass, and will produce smaller outputs,which are a measure of the overlap of those nontarget reagent spectrainto the filter signal. For example, the filter's output for the targetreagent might be 850, and the filter's output for the other 2 reagentsmight be 100 and 50, respectively. If the 3 reagents were added togetherin a single well, the total output would be 1000, and the proportionswould be 850:100:50. These coefficients are measured for each welllocation and filter separately, which gives a complete set ofcoefficients for simultaneous equations. This will allow a solution forany combination of concentrations of reagent in one sample well. Furtherin the preferred embodiment, these coefficients will also be normalizedby the total intensity read in the “total emission” filter, so that thecalculation will result in the same intensity as the instrument wouldmeasure if only a single reagent was measured by the “total emission”filter. This calculation may be shown as follows for a simple case ofblue and green reagents (abbreviated as R in the calculations), and blueand green and total emission filters (abbreviated as F in thecalculations):Let A=(output of the instrument for blue R thru the blue F)/(output ofinstrument for blue R thru total emission F);Let B=(output of the instrument for green R thru the blue F)/(output ofinstrument for green R thru total emission F);Let C=(output of the instrument for blue R thru the green F)/(output ofinstrument for blue R thru total emission F);Let D=(output of the instrument for green R thru the green F)/(output ofinstrument for green R thru total emission F);These coefficients are measured for each well prior to running amulti-color run.Then for a multi-reagent/color run,(output of the instrument for the blue F)=A×(true intensity of blueR)+B×(intensity of green R); and(output of the instrument for the green F)=C×(true intensity of blueR)+D×(intensity of green R)

These 2 simultaneous equations are then solved for the true intensity ofthe blue and green reagents by the processing software, under control ofprocessor 50.

Further in step G, the raw output of the instrument for each filter isnormalized for integration time before solving the equations.

The resulting intensities could then be calibrated as concentration byuse of standards as described in the previous section.

Finally, in step H, the analyzed data is presented in a user-acceptableformat, again controlled by processor 50.

The invention may be further understood by reference to examples ofassays practiced in HTS format, demonstrating the dynamic range andflexibility of the NorthStar™ luminometer.

EXAMPLES Example 1 Purified cAMP Quantitation

cAMP standards were serial diluted and added to a 96-well assay platewith alkaline phosphatase conjugated cAMP and anti-cAMP. Plates wereprocessed with the cAMP-Screen™ protocol and imaged for 1 minute on theNorthStar™ 30 minutes after addition of CSPD®/Sapphire-II™. Asensitivity of 0.06 pM of purified cAMP is achieved with cAMP-Screen™ onthe NorthStar™ workstation. The results are depicted in FIG. 8.

Example 2 cAMP Induction in Adrenergic β2 Receptor-Expressing C2 Cells

Adrenergic β2 Receptor-expressing C2 cells were plated in a 96-wellplate (10,000 cells/well) and stimulated with isoproterenol for 10minutes. cAMP production was quantitated in cell lysates using thecAMP-Screen™ assay. The assay plate was imaged for 1 minute on theNorthStar™, 30 minutes after addition of CSPD®/Sapphire-II™. IncreasingcAMP levels were detected on the NorthStar™ from the stimulatedadrenergic receptor. The results are depicted in FIG. 9.

Example 3 Luc-Screen™ Reporter Gene Assay in 96-, 384- and 1,536-wellFormat

pCRE-Luc-Transfected cells were seeded in 96-, 384- and 1,536-wellplates, incubated for 20 hours with forskolin, and assayed with theLuc-Screen™ system. PCRE-Luc contains the luciferase reporter gene underthe control of a cAMP response element (CRE). Forskolin inducesintracellular cAMP production through the irreversible activation ofadenylate cyclase. All plate formats demonstrate comparableforskolin-induced cAMP levels. The results are depicted in FIG. 10.

Example 4 Forskolin Induction of pCRE-Luc Transfected NIH-3T3 Cells

pCRE-Luc-Transfected cells were seeded in a 96-well plate. Four randomwells were induced for 17 hours with 1 mM forskolin and the entire platewas assayed with the Luc-Screen™ system. The results are shown in FIG.11.

Example 5 Dual-Light® Quantitation of Luciferase & β-GalactosidaseReporter Enzymes

NIH/3T3 cells were co-transfected with pCRE-Luc and pβgal-Control, andseeded into a 96-well microplate (2×10⁴ cells/well). Cells wereincubated with forskolin for 17 hours. Modified Dual-Light® Buffer A wasadded to cells and incubated for 10 minutes. Modified Dual Light® BufferB was injected and luciferase-catalyzed light emission was measuredimmediately. Thirty minutes later, Accelerator-II was added, and thenβ-galactosidase-catalyzed light emission was quantitated on theNorthStar™ HTS workstation. Quantitation is shown graphically in FIG.12.

Example 6 Normalized Fold Induction of Luciferase Reporter

Fold induction of luciferase activity was calculated followingnormalization to β-galactosidase activity. The Dual-Light® assay enablesthe use of a control reporter for normalization, or to monitornon-specific effects on gene expression. This is depicted in FIG. 13.

Example 7 Effect of BAPTA-AM on Antagonist Activity

CHO-Aeq-5HT2B cells were loaded with coelenterazine h+/−0.5 μM BAPTA-AMfor 4 hours. The antagonist methysergide was added to the charged cellsfor 30 minutes. 1 μM agonist a-Me-5HT was injected, and the emittedlight was integrated for 20 seconds on the NorthStar™ system. Thereported 1050 for methysergide (0.6 nM) is unchanged in the presence ofBAPTA-AM. The data obtained appears in FIG. 14.

Example 8 Effect of BAPTA-AM on Peptide Agonist Stimulated of the Orexin2 Receptor

CHO-Aeq-OX2-A2 cells (Euroscreen) were loaded with coelenterazineh+/−0.6 μM BAPTA-AM for 4 hours. The peptide agonist Orexin B wasinjected into the wells, and the emitted light was integrated for 20seconds on the NorthStar™. Using this assay on the NorthStar™ system,the reported EC50 for Orexin B (0.75 nM) is unchanged in the presence ofBAPTA-AM. This is shown in FIG. 15.

This invention has been described generically, by reference to specificembodiments and by example. Unless so indicated, no embodiment orexample is intended to be limiting. Alternatives will occur to those ofordinary skill in the art without the exercise of inventive skill, andwithin the scope of the claims set forth below.

What is claimed is:
 1. A luminescence detecting apparatus, comprising: aphotosensitive detector; a sample chamber configured for placement of aplurality of wells containing respective luminescent samples; asubstrate comprising a two-dimensional array of openings and positionedduring use between the plurality of wells and the detector, theplurality of wells aligned during use to respective ones of thecorresponding array of openings, the array of openings configured tosimultaneously pass emissions from at least some of the wells to thephotosensitive detector and to block some of the emissions from beingreceived by the photosensitive detector; a lens disposed along anoptical path between the two-dimensional array of openings and thedetector; and wherein the two-dimensional array of openings is disposedalong an optical axis thereof, the only light from the sample wellsreceived by the detector during use is light from the sample wells thatis parallel or semi-parallel to the optical axis.
 2. The apparatus ofclaim 1, wherein the apparatus comprises a luminometer.
 3. The apparatusof claim 1, further comprising a central processing unit for controllingthe analysis of the plurality of luminescent samples.
 4. The apparatusof claim 1, further comprising a defogger configured to preventcondensation.
 5. The apparatus of claim 1, further comprising a heaterdisposed below the plurality of wells during use.
 6. The apparatus ofclaim 1, further comprising a plurality of wells, wherein the detectoris configured to sequentially detect light at different wavelengthsemitted from the plurality of wells.
 7. The apparatus of claim 1,further comprising a plurality of luminescent samples disposed withinrespective ones of the plurality of wells during use.
 8. The apparatusof claim 7, wherein each of the plurality of luminescent samples is oneof a bioluminescent material or a chemiluminescent material.
 9. Theapparatus of claim 1, further comprising a plurality of wells, whereinthe wells are disposed on a plate.
 10. The apparatus of claim 1, furthercomprising a filter disposed along the optical path between thetwo-dimensional array of openings and the detector, the filterconfigured to permit passage of a specific wavelength of light emittedfrom the wells.
 11. The apparatus of claim 1, wherein thetwo-dimensional array of openings is in close proximity to the pluralityof wells during use, so as to enhance restriction of stray light fromthe wells.
 12. The apparatus of claim 1, wherein the lens comprises atleast one of a Fresnel lens, an aspheric lens, or a molded plastic lens.13. The apparatus of claim 1, wherein: the detector comprises a chargecoupled device (CCD); the lens is a primary lens and the apparatusfurther comprises a camera lens disposed between the primary lens andthe CCD; and the camera lens is configured to simultaneously image theluminescent samples of more than one of wells.
 14. The apparatus ofclaim 1, further comprising a temperature controller, wherein the samplechamber is temperature controlled for elevating the sample chamber abovean ambient temperature.
 15. The apparatus of claim 1, wherein theplurality of wells are in simultaneous optical communication with thephotosensitive detector, and wherein the photosensitive detector and thetwo-dimensional array of openings together are configured tosimultaneously detect the plurality of respective luminescent samplescontained in the plurality of wells.
 16. The apparatus of claim 14,wherein the sample chamber comprises a bottom surface and the substrateis positioned during use above the sample chamber.
 17. A method foranalyzing a plurality of luminescent samples, comprising the steps of:placing a plurality of luminescent samples in a respective plurality ofsample wells; placing the plurality of sample wells in a luminescencedetecting apparatus, the luminescence detecting apparatus comprising aplurality of photosensitive detector elements, a first lens, and asubstrate comprising a two-dimensional array of openings; registeringthe plurality of sample wells to respective openings of thetwo-dimensional array of openings; passing emissions from at least someof the wells through the two-dimensional array of openings to respectiveones of the plurality of photosensitive detector elements; using thesubstrate to prevent some of the emissions from the at least some of thewells from being received by the photosensitive detector elements;wherein the two-dimensional array of openings is disposed along aplurality of optical paths between at least two of the sample wells andrespective photosensitive detector elements of the plurality ofphotosensitive detector elements; wherein the first lens is disposedalong at least one of the optical paths at a location between thesubstrate and the detector; and wherein the substrate is configured suchthat the only light from the sample wells received by the detector islight that is parallel or semi-parallel to an optical axis of firstlens.
 18. The method of claim 17, further comprising positioning asecond lens disposed along the optical path at a location between thefirst lens and the detector.
 19. The method of claim 17, wherein thesample chamber comprises a bottom surface and the substrate ispositioned during use above the sample chamber.
 20. A luminescencedetecting apparatus, comprising: a detector comprising a plurality ofphotosensitive elements; a sample chamber configured for placement of aplurality of wells containing respective luminescent samples; asubstrate comprising a two-dimensional array of openings positionedduring use between the plurality of wells and the photosensitiveelements, the array of openings configured to simultaneously passemissions from at least some of the wells to respective photosensitiveelements of the detector and to block some of the emissions from beingreceived by the respective photosensitive elements; a lens disposedalong an optical path between an opening of the substrate and arespective photosensitive element of the detector; and a heater disposedduring use below the sample chamber.
 21. The apparatus of claim 20,further comprising a defogger configured to prevent condensation. 22.The apparatus of claim 20, further comprising a plurality of samplewells disposed within the sample chamber and aligned to correspondingopenings of the array of openings.
 23. The apparatus of claim 20,wherein the two-dimensional array of openings is disposed along anoptical axis thereof, the only light from the sample wells received bythe detector is light from the sample wells that is parallel orsemi-parallel to the optical axis.
 24. The apparatus of claim 20,wherein the detector is configured to sequentially detect light atdifferent wavelengths emitted from the plurality of sample wells. 25.The apparatus of claim 20, wherein each of the plurality of luminescentsamples is one of a bioluminescent material or a chemiluminescentmaterial.
 26. The apparatus of claim 20, further comprising a filterdisposed along the optical path between the two-dimensional array ofopenings and the detector, the filter configured to permit passage of aspecific wavelength of light emitted from the sample wells.
 27. Theapparatus of claim 20, wherein the two-dimensional array of openings isin close proximity to the plurality of sample wells so as to enhancerestriction of stray light from the sample wells.
 28. The apparatus ofclaim 20, wherein: the detector comprises a charge coupled device (CCD);the at least one lens comprises a primary lens and secondary lensdisposed between the primary lens and the CCD; and the secondary lens isconfigured to simultaneously image the luminescent samples of more thanone of wells.
 29. The apparatus of claim 20, further comprising atemperature controller, wherein the sample chamber is temperaturecontrolled for elevating the sample chamber above an ambienttemperature.
 30. The apparatus of claim 20, wherein the plurality ofwells are in simultaneous optical communication with the photosensitivedetector, and wherein the photosensitive detector and thetwo-dimensional array of openings together are configured tosimultaneously detect the plurality of respective luminescent samplescontained in the plurality of wells.
 31. The apparatus of claim 20,further comprising a plurality of wells containing respectiveluminescent samples, wherein the array of openings simultaneously passemissions from at least some of the wells to respective photosensitiveelements of the detector and block some of the emissions from beingreceived by the respective photosensitive elements.
 32. The apparatus ofclaim 20, wherein the sample chamber comprises a bottom surface and thesubstrate is positioned during use above the sample chamber.